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Team:Evry/Protocols/09
From 2013.igem.org
Siderophore detection
Aim
Once our bacteria is transformed with the plasmid with the Fur Binding Site and Lac I and the two plasmids with Enterobactins genes, we need to check if our system really work. The production of siderophores can be detected visualy with Blue Agar Chrome Azurol S (CAS). Without siderophore in the medium, CAS and Hexadecyltrimethylammonium bromide (HDTMA)complexes with ferric iron, producing a blue color. When a bacteria strain produce siderophore, the medium color change from blue to orange.
Preparation
Protocol adapted from Louden, Haarmann, and Lynne, “Use of Blue Agar CAS Assay for Siderophore Detection.”Blue Dye
Solution 1Dissolve 0,06 g of CAS in 50 mL of distilled water.
Solution 2
Dissolve 0,0027 g of FeCl3-6H20 in 10 mL of 10 mM HCl.
Solution 3
Dissolve 0,073 g of HDTMA in 40 mL of distilled water.
Mix
Mix solution 1 with 9 mL of solution 2. Then mix with solution 3.
Solution should have a blue color.
Autoclave and store in a bottle.
Mixture solution
Minimal Media 9 (MM9) Salt Solution StockDissolve 15 g of KH2PO4, 25 g of NaCl and 50 g of NH4Cl in 500 mL of distilled water.
NaOH Stock
Dissolve 25 g of NaOH in 150 mL of distilled water.
pH should be around 12.
20% Glucose Stock
Dissolve 20 g of glucose in 100 mL of distilled water.
Casamino Acid Solution
Dissolve 3 g of Casamino acid in 27 mL of distilled water.
Extract with 3% 8-hydroxyquinoline in chloroform to remove iron.
Filter with a 0,22 µm millipore.
CAS agar preparation
Site developped by Gabriel Guillocheau
